Flow Cytometry Marta Argenti, PhD student Department of Biomedical Sciences Padua 14.12.12

Flow ~ cells in motion

Cyto ~ cell

Metry ~ measure

Flow Cytometry is the measurement

of cells in a flow system

Physical properties:

- size - granularity, internal complexity Chemical properties:

- fluorescence intensity (fluorescent probes and Ab)

Uses of Flow Cytometry Immunophenotyping

DNA cell cycle/tumor ploidy

Membrane potential

Ion flux

Cell viability

Intracellular protein staining

pH changes

Cell tracking and proliferation

Sorting

Redox state

Chromatin structure

Total protein

Lipids

Surface charge

Membrane fusion/runover

Enzyme activity

Oxidative metabolism

Sulfhydryl groups/glutathione

DNA synthesis

DNA degradation

Gene expression .... and many others

To measure large numbers of single cells within a short period of time (tens of seconds to minutes).

Simultaneous collection of the most diverse parameters

of each cell.

Reproducibility and statistical reliability.

The heterogeneity of populations can be revealed and different subsets of cells identified and quantified.

Selected cell populations can also be physically sorted for further study.

It requires a suspension of single cells.

A flow cytometer need to combine:

Fluidics Introduces and focuses the cells in suspension for“interrogation”.

Optics Generates and collects light signals.

Electronics Changes optical signals to electronic signals and digitize for computer analysis.

The Fluidics System

Hydrodynamic focusing

The cells flow one-by-one into the cytometer to do single cell analysis.

Becton Dickinson FACSAria

sample

flow cell

A flow cytometer need to combine:

Fluidics Introduces and focuses the cells in suspension for“interrogation”.

Optics Generates and collects light signals.

Electronics Changes optical signals to electronic signals and digitize for computer analysis.

L A S E R

Fluorescence /

180°

90°

Optics cells

L A S E R

Fluorescence /

180° 90°

Forward Scatter diffracted light, it is a measure of CELL SIZE (FSC) Side Scatter refracted and reflected light, it is a measure of (SSC) GRANULARITY AND INTERNAL COMPLEXITY

L A

S E

R

cell

Optics

Fluorescence / Side Scatter

Forward Scatter

Optics FL1

Red Laser 635 nm

SSC

FL3

FL4

670LP

661/16

585/42

488/10

90/10 Beam Splitter

DM 560SP

Fluorescence

Collection Lens

DM 640LP

Half Mirror

488/10

Blue Laser

488 nm Focusing

Lens

FL2

530/30

Beam Combiner

FSC

(FACScalibur 4 colours) sample

. Flow Cell

A flow cytometer need to combine:

Fluidics Introduces and focuses the cells in suspension for“interrogation”.

Optics Generates and collects light signals.

Electronics Changes optical signals to electronic signals and digitize for computer analysis.

Creation of a voltage pulse

LASER

cell

Detector = Photomultiplier tube

Photomultiplier tubes collect photons of light and convert them to current.

pulse height

The Analog-to-Digital Converter assigns a digital value to the voltage pulse.

Displaying flow cytometry data:

Histogram 1 parameter

Forward Scatter

size cell n

umber

Dot Plot example: human peripheral blood leucocytes

2 parameters

FSC

SSC

Forward Scatter

Sid

e S

catt

er

Lymphocytes

Monocytes

Granulocytes

debris, dead cells

size

granu

larity

Contour Plot

Greyscale Density Density Plot

Isometric Plot

3D Plot

Other cytograms

Fluorescence

L A S E R

Fluorescence /

180° 90°

absorption

emission

Fluorochromes

1. Covalently bound to other probes: ANTIBODIES

2. Non covalently bound: fluorescent dyes which bind

to DNA, dyes sensitive to pH, Ca(II)...

Cell Death Analysis:

Propidium iodide (PI) binds to DNA and it isn't cell permeant

Forward Scatter

Sid

e S

catt

er

Pro

pid

ium

Iod

ide

FITC

Propidium Iodide

Co

un

t

dead cells

living cells

living cells

dead cells 11.7%

Reactive Oxygen Species production:

Change in dichlorofluorescein fluorescence of mouse striatal synaptosomes after 1-h incubation at 37 °C alone or with MDMA. (Chipana et al. Neuropharm 51 (4), 2006)

Forward Scatter Forward Scatter Forward Scatter Forward Scatter

Gre

en

Flu

ore

sce

nce

Gre

en

Flu

ore

sce

nce

CTRL MDMA

DCF

2'7'-dichlorofluorescin (DCFH) 2'7'-dichlorofluorescein (DCF)

ROS

Green Fluorescence

Forward Scatter

Sid

e S

catt

er

Reticulocyte Analysis: anaemia classification

bind to RNA

Human peripheral blood reticulocytes

RNA

reticulocyte erytrocyte

reticulocytes + erytrocyte

platelets

erytrocytes

Immunophenotyping:

The Cluster of Differentiation is a protocol used for the identification of cell surface molecules present on LEUKOCYTES

To evaluate individual cells for the presence and absence of specific antigens

Immunophenotyping: analysis of leukaemias and lymphomas submandibular gland biopsy

A leukaemia or lymphoma will express a specific set of markers depending on which stage and pathway of blood cell differentiation are affected.

B-cell lymphoid neoplasm

CD5 + CD19 weak intensity FMC-7 + CD20 + kappa light chain + lambda light chain -

Mantle cell lymphoma

Immunophenotyping: HIV infection

HIV CD4+lymphocytes depletion CD4+ count to measure disease progression

leukocytes

Sid

e S

catt

er

CD4+lymphocytes

CD4+ lympohocyte

700 x 103 /mL

Sorting: to capture and collect cells of interest for further

analysis/use

FACS = Fluorescence-Activated Cell Sorting

Sorting: to capture and collect cells of interest for further

analysis/use

FACS = Fluorescence-Activated Cell Sorting

sperm sorting: sex selection (artificial insemination, in vitro fertilization)

bone marrow transplant

stem cells isolation

Sorting: some clinical applications

DNA Analysis:

• information about cells ploidy • distribution of cells across the cell

cycle

G0 /

M /

Propidium Iodide is a red fluorescent molecule which binds to nucleic acids.

[Fine needle aspirate of a breast carcinoma] There are normal, diploid cells present together with an aneuploid TUMOUR.

Some fluorochromes used to label antibodies

tan

de

m d

yes

•

•

•

Gating

Data from human peripheral blood leucocytes:

Lymphocytes

FSC

SSC

Features of the apoptotic cascade that can be observed using flow cytometry: Expression of proteins involved in apoptosis (immunofluorescence) Activation of caspases (rhodamine 110 attached to caspase-specific peptide) Changes in the mitochondrial membrane potential (TMRM, DiOC6, rhodamine 123, JC-1) Changes in the plasma membrane (PI, 7-AAD, Annexin V) Cell shrinkage (Scatter Plot) Chromatin changes DNA degradation (PI, TUNEL assay)

Immature human thymocytes incubated with dexamethasone 7-AAD = 7-aminoactinomycin D

An example of multiparametric analysis:

Others applications: Intracellular calcium ions Fluo-3, Fura Red

Intracellular pH Carboxy SNARF®-1

Intracellular glutathione Monobromobimane give a fluorescent conjugate with glutathione

Drug uptake RNA content Acridine orange, Pyronin Y

Cell proliferation 5’-bromodeoxyuridine (BrdUrd) is incorporated into the DNA in place of thymidine.

Forward Scatter

Sid

e S

catt

er

ANNEXIN V

Cell Death Analysis:

Propidium iodide (PI) marks cells with broken cell membrane Annexin V mark apoptotic cells

UV-B radiation-induced apoptosis in Jurkat cells

Creation of a voltage pulse

LASER

cell

Photomultiplier tube

Photomultiplier tubes

collect photons of light and convert them to current.

pulse height

The Analog-to-Digital Converter assigns a digital value to the voltage pulse

LASER

Event # 1

FL1 FL2 FL3 FL4 SSC

A bivariate flow karyotype of a normal human female.

Hoechst 33258 binds to AT-rich regions chromomycin A3 binds to GC-rich regions

chromomycin A3

Chromosome analysis and sorting

DNA Analysis: karyotyping

(a) very small samples can be analyzed (5,000–10,000 mitochondria) (b) preparations contaminated with other cellular constituents can be handled (using mitochondria-specific markers) (c) potential identification of mitochondrial subpopulations (d) mitochondria can be sorted for further analysis of, e.g., protein profile.

Flow Cytometric Analysis of Isolated Mitochondria

NAO = 10-nonyl acridine orange

ROS

Δψm

Mitochondria swelling induces a decrease in Side Scatter

Compensation

Forward Scatter

Fluorescence / Side Scatter

Optics FL1

Red Laser 635 nm

SSC

FL3

FL4

670LP

661/16

585/42

488/10

90/10 Beam Splitter

DM 560SP

Fluorescence

Collection Lens

DM 640LP

Half Mirror

488/10

.

Blue Laser 488 nm

Focusing

Lens

FL2

530/30

Beam Combiner Flow Cell

FSC

(FACScalibur 4 colours) sample